Normal diploid human cells have a limited capacity to proliferate, a process termed cellular senescence. Increasing evidence over the last decade has implicated telomeres, the structures that cap the ends of the chromosomes as the molecular clock that counts the number of times the cell has divided. The mechanism of lagging strand DNA synthesis prevents DNA polymerase from replicating the DNA all the way to the 5' end of a linear chromosome, leaving a 3' overhang and causing the chromosome to shorten very time a cell divides. The P.I. has developed a method for purifying telomeres (based on the presence of the 3' G-rich overhang) that produces a 1000-fold enrichment in a single step. This permits him to address the following Specific Aims: 1) Use telomeric sequence tags to determine telomere fate during proliferative life span; 2) Define the structure of the telomeric overhang in both normal diploid and immortal human cells; 3) Determine the contribution of Okazaki fragment size to telomere shortening; 4) Develop in vitro/in vivo model system to study the mechanisms regulating telomere shortening. Knowledge gained from these studies may lead to the ability to manipulate rates of telomere shortening, with consequences both for slowing cellular senescence and enhancing the efficacy of anti-telomerase cancer therapeutics.